Multi-copy <i>aiiA</i> genes encoding quorum-quenching enzymes in <i>Bacillus thuringiensis</i> : identification and functional characterization of the novel AHL-lactonase, AiiA2

Abstract Quorum sensing mediated by N-acylhomoserine lactones (AHLs) plays a key role in the regulation of virulence in many plant-pathogenic bacteria, and enzymatic degradation of AHLs represents a promising biocontrol strategy known as quorum quenching. The AHL lactonase gene aiiA is widely distributed within the genus Bacillus and is generally considered to be present as a single-copy gene. In this study, we show that specific strains of Bacillus thuringiensis harbor two distinct aiiA homologs. Genome analyses of environmental B. thuringiensis isolates, together with publicly available genome sequences, revealed a phylogenetically distinct aiiA homolog in addition to the canonical gene. Phylogenetic analysis classified these homologs into two groups, designated AiiA1 and AiiA2. Comparative genomic analysis indicated that aiiA2 is located within variable genomic regions, suggesting acquisition via horizontal gene transfer through mechanisms other than transposon-mediated transposition. Functional assays confirmed that both AiiA1 and AiiA2 possess AHL-degrading activity. Quantitative analyses showed that the specific activities of both enzymes increased with increasing temperature, and although AiiA2 exhibited slightly higher activity than AiiA1 across the tested temperature range, no dramatic difference in AHL-degrading activity was observed between the two enzymes. These findings highlight previously unrecognized diversity in quorum-quenching systems within B. thuringiensis and suggest that the coexistence of multiple AHL lactonases with largely comparable activities may contribute to a flexible and robust quorum-quenching capacity in plant-associated environments.